Certain aspects of the present invention generally relate to RNA molecules and the alternative splicing thereof. More specifically, certain aspects of the present invention describe precise exon selection in a multi-exon device.
Cells coordinate largely complex tasks, such as metabolic processes, differentiation, gene expression, and transport, by processing signals within the cellular environment or from external cues to elicit a specific genetic response. An average mammalian gene encodes approximately three RNA transcripts, which can then be translated to generate distinct protein isoforms (Pan et al, Nature Genetics 2008, 40: 1413-1415). Typically natural proteins like densin, a LAP protein, modulate their usage of protein-protein interaction and localization domains to generate diverse and functionally distinct proteins with unique roles within their cellular networks (Jiao et al, J Neurochem 2008 105: 1746). While a number of engineered molecular platforms and devices for mammalian cells have been constructed, they are primarily limited to controlling gene expression, that is increasing or decreasing gene expression (i.e. turning a gene “ON” or “OFF”) or modulating protein activity post-translationally once the protein isoform has been produced. Our ability to effectively engineer biological systems is limited by the tools and strategies available to detect, transmit, and so control molecular information. Devices that support more sophisticated control, such as control of spatial organization or protein function, are needed to advance the scale and complexity with which mammalian devices can be designed and integrated within native cellular networks.
RNA-based control devices have been previously developed to process biomolecular inputs and produce regulated protein outputs (Liang et al Mol Cell 2011 43: 915-926; Culler et al. Science 2010 330: 1251-1255). RNA exhibits unique advantages as a substrate for genetic device design because RNA structures can be designed with relative ease and RNA exhibits diverse sensing and regulatory activities. In one example, RNA devices based on an alternative splicing mechanism linked disease biomarkers to cell death by modulating the inclusion of a premature stop codon in a suicide gene (Culler et al, Science 2010 330: 1251-1255). Alternative splicing, a prevalent post-transcriptional regulatory mechanism, is a process by which multiple protein isoforms are generated by altering the ways in which exons, or protein coding regions, are joined, and introns, or non-protein coding regions, are excised. While alternative splicing has the capacity to decompress information encoded in a single gene and modulate the usage of domains, this capability has not been harnessed in engineered molecular systems.
Studies performed using high-throughput sequencing technology estimate approximately 95% of human multi-exon genes undergo alternative splicing, with an average of three unique transcripts encoded per gene (Pan et al, Nature Genetics 2008 40: 1413-1415). In this manner, alternative splicing is critical for increasing protein diversity in natural systems. Yet prior studies have largely focused on linking alternative splicing events to turning “ON” or “OFF” gene expression, rather than increasing protein diversity in the cell. Such designs have modulated exon skipping to modulate the inclusion of a premature stop codon (Culler et al, Science 2010 330: 1251-1255), the incorporation of frameshift mutations to decide which one of two genes downstream of the final exon is translated (Newman RNA 2006 12: 1129-1141), and intron excision in response to small molecule binding (Kim et al BMC Mol Biol 2008 9: 23). While these constructs depend on an alternative splicing event for the output, they are largely limited to controlling gene expression in the context of simple alternative splicing modes or affecting the translation of the mRNA molecule that is generated.